Axon pathfinding is an essential process required for the establishment of proper neuronal connections during development. Elucidating the mechanisms governing axon growth and guidance will be important not only for understanding the basic developmental processes by which the nervous system is established but may also shed light on certain pathological conditions arising from axon wiring defects. In fact, Dr. Elizabeth Engle's lab has identified several neurological disorders resulting from aberrant nerve connectivity, known as the congenital cranial dysinnervation disorders (CCDDs). In one of these disorders, congenital fibrosis of the extraocular muscles type 3 (CFEOM3), patients exhibit ocular motility defects due to failed cranial nerve innervation of the extraocular muscles. Eight different missense mutations underlying this disorder mapped to beta-tubulin isotype III (TUBB3), a highly dynamic, neuron-specific tubulin isoform whose expression is upregulated during the period of axon growth and pathfinding. Each mutation results in CFEOM3, but can also be accompanied by aberrant guidance of additional cranial, spinal, and central axons. A knock-in mouse model harboring the most common mutation identified in human CFEOM3 patients, and resulting in the R262C amino acid substitution, exhibits axon guidance defects without alterations in cortical architecture, suggesting this disease is primarily caused by aberrant axon connectivity. However, the mechanisms by which mutations in TUBB3 affect axonal microtubules and axon guidance have not yet been investigated. This training fellowship consists of two aims, the first of which proposes to establish a neuronal model system in order to investigate the effect of pathological mutations in TUBB3 on microtubule organization and dynamics in axons. I will create knock-in disease mutations in mouse embryonic stem (ES) cells through homologous recombination and then differentiate these ES cells into motor neurons. The localization and organization of microtubules in the growth cone will be analyzed by immunostaining and the stability and dynamics of microtubules in the axons will be investigated through a biochemical approach and live cell imaging. The second aim will utilize the ES cell-derived neuronal model system in order to determine the effect of the TUBB3 mutations on axon guidance. Specifically, growth cone collapse and axon turning in response to attractive and repulsive guidance cues will be investigated. Collectively, the results from these aims will lead t a better understanding of the role of microtubules in axon guidance, and, more importantly, will elucidate the mechanisms by which TUBB3 mutations result in human neurological disorders. PUBLIC HEALTH RELEVANCE: During development, neurons extend long processes known as axons, which are required to form precise connections to ensure proper nervous system function. Recently, mutations in the beta-III tubulin gene (TUBB3) have been shown to cause human neurological disorders by affecting the microtubule cytoskeleton and perturbing the guidance of axons to their appropriate synaptic targets. This proposal seeks to investigate the effects of these mutations on microtubule organization and dynamics in order to identify the developmental role of TUBB3 in axon guidance and, more importantly, to increase our understanding of human neurodevelopmental disorders resulting from abnormal circuit formation.